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ISSN 1995-6673 Pages 55 - 62 Jordan Journal of Biological Sciences

Evaluation of Hexaploid Wheat Varieties for Making Bread by High Molecular Weight (HMW) and Low Molecular Weight

University of Sulaimanya ah, Iraq

Abstract

ﺺﺨﻠﻤﻟا

(LMW) Analysis

Nawroz Abdul-razzak Tahir*

h, College of Agriculture

,

Bakrajo, Sulaimany

Knowledge of composition of high molecular weight glute

glutenin subunits (LMW-GS) an bread

impro bread Cham alleli electr prope bread small the g thous thous show wher differ subun Glu-B allele bread 7+9 w the v quali lowes was e molec (LMW

نا ﺔﻓﺮﻌﻣ ﻦﻴﻨﻴﺗﻮﻠﻐﻟا تاذ نازوﻻا ا ﺌﻳﺰﺠﻟ ﺔ ةﺮﻴﻐﺼﻟا و نازوﻻا ا ﻳﺰﺠﻟ ﺌ ﺔ

ا ﺮ

ﺔﻄ

ا

ﻢ ا

ة

, ا

ا ت ﺔ

ﻟ ﻲ ت

G G

و ﻓ

ﻊ ا

© 2009 Jordan Journal of Biological Sciences. All rights reserved

Keywords: Bread Wheat; Wheat Quality; HMW and LMW Of Glutenin; SDS-PAGE Electr

nin subunits (HMW-GS) and low molecular weight d their associations with making and quality will contribute to genetically ving processing quality of bread wheats in Iraq. Six wheat varieties (Tammuz, Aras, Rabia, Cham 4, 6 and Costantino) were conducted to detect the c variation at Glu-1 and Glu-3 loci by SDS-PAGE ophoresis and to understand their effects on dough rties. Important methods applied for the breeding of -quality wheat (Triticum aestivum L.) consist of -scale bread-quality tests for the determination of rain protein content, SDS-sedimentation volume, and kernel weight and kernel diameter. The and kernel weight and SDS-sedimentation volume ed relatively higher significant among the varieties, eas the flour yield showed no significance

ence. The results of SDS-PAGE indicate that its/alleles 1 and null at Glu-A1, 7, 20 and 7+9 at

1, 2+12 and 5+10 at Glu-D1, allele a at Glu-A3, s d and m at Glu-B3 and alleles j and k at Glu-D3 in wheat varieties. The lowest frequency of subunit as found in variety Tammuz. On the other hand, ariety Tammuz showed the highest value of score ty and the varieties Cham 4 and Cham 6 had the t value of score quality. Genetic diversity of wheat valuated by constructing the dendrogram for high ular weight (HMW) and low molecular weight

) gluten subunit bands.

ةﺮﻴﺒﻜﻟا و ﺎﻬﺗﺎﻃﺎﺒﺗرا ﻊﻣ ﺔﻋﺎﻨﺻ ﻴﻋﻮﻧو ﻒﻴﻏﺮﻟا ﺘ

ﻲﻓ ﻢﻬﺴﺘﺳ ﺔ ﺔﺠﻟﺎﻌﻤﻟا

ﺎﻴﺛارو ﻦﻴﺴﺤﺘﻟ ﺔﻴﻋﻮﻧ ﺰﺒﺨﻟا ﺔﻄﻨﺤﻟاو ﻓ ﻲ قاﺮﻌﻟا . ﺪﻗ و ا ﺖﻣﺪﺨﺘﺳ

ﺔﻘﻳﺮﻃ دﺪﻌﺘﻣ مﻮﻳدﻮﺼﻟا ﻞﻜﻳدود تﺎﺘﻳﺮﺒآ مﻼﻬﻟ ﻲﺋﺎﺑﺮﻬﻜﻟا ﻞﺼﻔﻟا

ﺪﻳﺎﻣﻼﻳﺮآﻷ ﻟ

ﻦﻴﻨﻴﺗﻮﻠﻐﻟا تﺎﺌﻳﺰﺟ ﻞﺼﻔ تاذ

نزﻮﻟا ا ﺌﻳﺰﺠﻟ ﻲ ﻴﻐﺼﻟا

نزﻮﻟاو ا ﻳﺰﺠﻟ ﻲﺌ ﺮﻴﺒﻜﻟا و ﻦﻋ ﻒﺸﻜﻠﻟ ﻦﻳﺎﺒﺗ

تﻼﻴﻟﻷا ﻊﻗاﻮﻤﻟا ﻰﻓ

Glu-1

و

Glu-3

ﻢﻬﻓو ﺎهﺮﻴﺛﺄﺗ ﻰﻠﻋ تﺎﻔﺻ ﻦﻴﺠﻌﻟا ﻓ ﻲ ﻒﻠﺘﺨﻣ فﺎﻨﺻا ﻨﺤﻟا

ﺔﻤﻋﺎﻨﻟ :

،زﻮﻤﺗ ﺁ سار ، ﺔﻌﻴﺑر مﺎﺷ ، 4

,

مﺎﺷ 6 و ﻮﻨﻴﺘﻧﺎﺘﺳﻮآ . نا أ

ه تارﺎﺒﺘﺧا ﻲه ﺔﻄﻨﺤﻟ ا و ﺰﺒﺨﻟا ﺔﻴﻋﻮﻧ ﺔﺳارﺪ ﻟ ﺔﻌﺒﺘﻤﻟا ﺐﻴﻟﺎﺳﻷ دﻮﺠﻟا

ا ىﻮﺘﺤﻣ ﺪﻳﺪﺤﺘ تﺎﺒﺳﺮﺘﻟا ﻢﺠﺣ ،ﻦﻴﺗوﺮﺒﻟ

ﻦﻴﺗوﺮﺒﻠﻟ ، نزو ﻒﻟﻷا

ﺔﺒﺣ ﻴﺑ ﺔﻳﻮﻨﻌﻣ قوﺮﻓ تزﺮﺑ ﺞﺋﺎﺘﻨﻟا نا . ﺔﺒﺤﻟا ﺮﻄﻗو , ﺞﺗﺎﻨﻟا ﻦﻴﺤﻄﻟ فﺎﻨﺻﻻ ﺔﺒﺴﻨﻟﺎﺑ ﺔﻄﻨﺤﻟا ﻦﻣ نزﻮﻟ

ﻒﻟﻷا ﺔﺒﺣ ﺎﺒﺳﺮﺘﻟا ﻢﺠﺣو

ﻦﻴﺗوﺮﺒﻠﻟ ، ﻓ ﻲ ﻦﻴﺣ ﺮﻬﻈﺗ ﻢﻟ ا ي ﺮﻔﻟا و ﻗ تﺎ ﻳﻮﻨﻌﻣ ﺔ ﺒﺴﻨﻟﺎﺑ فﺎﻨﺻﻻا ﻦﻴﺑ

ﻦﻴﺤﻄﻠ ﺞﺗﺎﻨﻟا إ ﺞﺋﺎﺘﻨﻟا ﺮﻴﺸﺗو

.

ﻰﻟ

دﻮﺟو ا ﻷ ﻼﻴﻟ ن 1 و غﻻ

(Null)

ﻓ ﻷ أ و

Glu-B1

ﻊﻗﻮﻣ ﻲ ﻓ 9 + 7

,

20

,

7 ت ﻼﻴﻟ ﻷ ا و

Glu-A1

ﻊﻗﻮﻣ ﻼﻴﻟ

و

12+2

5 + 10 ﻓ ﻲ ﻊﻗﻮﻣ

Glu-D1

ﻞﻴﻟﻷاو

a

ﻲﻓ ﻊﻗﻮﻣ

lu-A3

ﻼﻴﻟﻷاو ن و

d

m

ﻓ ﻲ ﻊﻗﻮﻣ

Glu-B3

ﻼﻴﻟﻷاو ن و

j

k

ﻲ ﻊﻗﻮﻣ

lu-

.

D3

ﻦﻴﻨﻴﺗﻮﻠﻐﻟا ناو ذ

و ﻞﻴﻟﻷا 7 + 9 ﺮﺜﻋ ﺎﻬﻴﻠﻋ ﻓ ﻂﻘ ﻓ ﻲ ﻒﻨﺼﻟا زﻮﻤﺗ

. ﻓ ﺰﺒﺨﻟا ﻊﻴﻨﺼﺗ ﻲ ﻓ ةدﻮﺠﻠﻟ ﺔﻤﻴﻗ ﻰﻠﻋأ زﻮﻤﺗ ﻒﻨﺼﻟا ﺮﻬﻇا ، ﻞﺑﺎﻘﻤﻟا ﻲ ﻦﻴﺣ ﻋأ ﺖﻄ مﺎﺷ فﺎﻨﺻﻻا 4

مﺎﺷو 6 ﻰﻧدأ ﺔﻤﻴﻗ ﻟ ةدﻮﺠﻠ ﻰﻓ

ﻴﻨﺼﺗ ﻋ سرد ﻦﻴﻨﻴﺗﻮﻠﻐﻟا ﺚﻴﺣ ﻦﻣ ﺔﻄﻨﺤ ﻠﻟ ﻲﺛارﻮﻟا فﻼﺘﺧ ﻻا نا ﺎﻤآ . ﺰﺒﺨﻟ يﺮﺠﺸﻟا ﻢﺳﺮﻟا ﻦﻳﻮﻜﺗ ﻖﻳﺮﻃ .

(Sodium Dodecyl Sulfate Polyacrylamide Gel ophoresis).

1. Introduction *

Wheat grain storage proteins are composed of 2 major fractions

high- (LMW GS) a

, gliadin and glutenin. Glutenin consists of both molecular-weight (HMW) and low-molecular weight ) subunits. The HMW-glutenin subunits (HMW- re encoded by Glu-A1, Glu-B1, and Glu- D1 on the

* Corr

long arm of chromosomes 1A, 1B and 1D, respectively (P

G

short arm of these chromosomes (Gupta et al., 1990).

G B S su b p all esponding author. nawrozbiology@gmail.com. d

ayne et al., 1980). The LMW-glutenin subunits (LMW- S) are encoded by Glu-A3, Glu-B3, and Glu-D3 on the lutenin subunits were also classified into A (HMW-GS), and C (LMW-GSs) subunits based on their mobility in DS-PAGE analysis (Gupta et al., 1990). These glutenin bunits are polymerized by intermolecular disulfide onds, which play a major role in the rheological roperties of wheat flour doughs. It has been shown that

elic variations of HMW-GSs and LMW-GSs affect ough properties in various wheat cultivars (Gupta et al.,

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© 2009 Jordan Journal of Biological Sciences. All rights reserved - Volume 2, Number 2 56

1991; Gupta et al., 1994; Khelifi et al., 1992; Nagamine et al., 2000; Payne et al., 1987a; Payne et al., 1987b). The HMW glutenins represents 5-10% of the total grain protein. The HMW glutenins are further subdivided into alleli

each quali much becau mobi

W of br impo on th LMW 60%

glute influe aggre cyste two d exten forma have al., 1 streng The decre termi propo shifti HMW 2001 polym is go streng glute and b (Ciaf 1998 exped increa traits traits

G exped deter of so diver can morp mark under glute

2. Materials and Methods

depar whea 6) so

origin of Tammuz, Aras and Rabia is Iraq while the origin of Cham 4 and Cham 6 is Syria (ICARDA). Costantino was used as a reference in this study. Cham 4 and Cham 6 were introduced to Iraq by FAO (Food and Agriculture O

2 2

W-GS was analyzed by yl sulfate polyacrylamide ins (30 gram) were ground to Th

2 3 m at fo p co sa 8 co st ro el at st C w 2

) was added to 500 μl 50%

r 30 min with agitation every 10 m

rp p 1 d ti so 2 w 2 an b µ g b S 1 (p te ap 1 (w B (A

w al c pairs on 1B and 1D and a single subunit on 1A and

of these subunits influences wheat flour and dough ty. The role of individual LMW-GSs is, however, less well characterized than that of HMW-GSs, se large numbers of the subunits display similar lities in SDS-PAGE analysis.

hile the HMW glutenins are the major determinants ead quality, LMW glutenins and gliadins are also rtant. Genes encoding the LMW glutenins are present e short arm of chromosome 1A, 1B, and 1D.The

glutenins are one-third of the total seed protein and of the total glutenins. The HMW and the LMW nins form extensive disulphide linked polymers that nce the dough quality. The LMW glutenins form gates may be important for dough strength. The ine residues in the LMW structure helps to separate

ifferent HMW polymer-building subunits. The chain ders (having two or more cysteine residue) allow the tion of stronger dough’s, while chain terminators the opposite effect (Greenfield et al., 1998; Masci et 998). The chain extender proteins have increased

th and stability due to the longer repetitive domains.

polypeptides with single cysteine residue have ased dough strength and stability as they act as chain nators in the glutenin polymers. The reduction in

rtion of LMW glutenins, results in dough properties ng towards greater strength due to an increase in the /LMW glutenin ratio (MacRitchie and Lafiandra

; Lawerence et al., 1998). The increase in the eric proteins results in a stronger dough strength that od for bread quality. In contrast the dough mixing th is reduced in deletion lines missing the HMW nins. An increase in the amount of polymeric protein etter flour performance has also been demonstrated fi et al., 1995; Rogers et al., 1997; Lafiandra et al., ). The use of registered crop varieties makes their itious identification important; its significance is sed by the diversity of varieties in many important . Each variety is characterized by a specific set of

that determine its use.

liadins and glutens are genetic markers allowing the itious and objective identification of a variety, mination of its genetic constitution, and determination

me important characteristics and traits. Genetic sity is the basis for successful crop improvement and

be estimated by different methods such as hological traits, end-use quality traits, and molecular ers (Fufa et al., 2005). The present study was

taken to evaluate the quality and genetic diversity in n-subunits in six wheat varieties using SDS-PAGE.

2.1. Plant Sample

Grains of wheat varieties were collected from the tment of Agriculture of Sulaimanyah. The bread t varieties (Tammuz, Aras, Rabia, Cham 4 and Cham wn, grown and harvested in the same location. The

rganization) since 1997.

.2. SDS-PAGE Electrophoresis .2.1. HMW-GS Extraction

The grain protein HM using SDS-PAGE (sodium dodec gel electrophoresis). The gra

fine powder and 20 mg was weighed in 1.5 ml microtube.

ree hundred microliter of protein extraction buffer [ 8.5% sample buffer (7% SDS, Tris-HCl 0.01 M (pH 6.8), 0% glycerol, 0.001% Coomassie bleu), 5% 2-

ercaptoethanol] was added to each micro tube, kept 2 hr room temperature (27°C) and centrifuged at 13000 rpm

r 10 min. The supernatant contains dissolved extracted rotein HMW-GS ready for experiment purposes, which uld be kept for longer time at 4°C. Before the loading of mples on the SDS-PAGE gel, the samples were heated at 0°C for 20 min and then loaded on SDS-PAGE. The gel

nsisted of a 15% separating gel (pH 8.4), beneath a 3%

acking gel (pH 6.8). Electrophoresis was carried out at om temperature using a home-made vertical ectrophoresis apparatus, and the running was performed 15 mA/gel for 18 hours. After 18 hours, the gels were ained in 12.5% (w/v) trichloroacetic acid, 0.01% (w/v) oomassie Brilliant Blue R250 and distained with distilled ater (Akhtar et al., 1994).

.2.2. LMW-GS Extraction The flour (20 mg (v:v) propan-2-ol at 60°C fo

in at room temperature, then centrifuged at 13000 m for 10 min. The residue was washed with 500 μl 50%

ropan-2-ol for 30 min at 60 ºC. After centrifuging at 3000 rpm for 10 min, the supernatant was again iscarded. This step to remove gliadins was repeated three mes. Glutenin was then solubilized with 500 μl of

lution [50% (v:v) propan-2-ol, 0.08 M Tris-Hcl (pH 8.5), 0 mM dithiothretol] at 60°C for 30 min. The supernatant

as diluted with 1 volume of solution [50% (v:v) propan- -ol, 0.08 M Tris-HCl (pH 8.5), 40 mM 4-vinylpyridine],

d incubated for 3 hr at 60°C. Glutenin was precipitated y 1 ml acetone and the dried pellet was solubilized in 200 l of buffer [7% SDS, Tris-HCl 0.01 M (pH 6.8), 30%

lycerol, 0.0

2

% Bromophenol blue or 0.001% Coomassie leu]. Finally, 30 µl samples were loaded into the slots of DS-PAGE(Cherdouh et al., 2005). The gel consisted of a 5% separating gel (pH 8.4), beneath a 3% stacking gel H 6.8). Electrophoresis was carried out at room mperature using a home-made vertical electrophoresis

paratus, and the running was performed at15 mA/gel for 8 hours. After 18 hours, the gels were stained in 12.5%

/v) trichloroacetic acid, 0.01% (w/v) Coomassie rilliant Blue R250 and distained with distilled water

khtar et al., 1994).

2.2.3. Two Dimensional Gels A-PAGE X SDS-PAGE The two dimensional Acid-PAGE x SDS-PAGE as performed by the protocol as described by Pagne et ., 1984. After the first dimension A-PAGE (Acid

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polyacrylamide gel electrophoresis), the gels were cut into single strips and incubated for 15 min in 0.0625 M Tris- HCl (pH 6.8), 2% (w/v) SDS, 40% (w/v) glycerol. The strips were then loaded onto a SDS-PAGE gel prepared as descr

temp reach descr 2.3. Q

Pe

flour mbustion method using a nitrogen Method 38-12 (AACC Asso

the A 2.4. S

0.5 g The cylinder was

5 times (one per second) at the first minute a

again min a (one in 1L 88%

soluti times was s the co 10 to with 2.5. M

• Th We prepared a 300 gram of

, shrunken and

r each variety were scored and the presence

and a LSD using (Unw const

3. Results and Dis

large

index showed significant differences, while flour yield was not significant (Table 1). Costantino gave the lowest thousand kernel weight and Aras gave the highest value. In terms of kernel quality, inverse relationships have been re

(O k w v C la th 8 A fi si h p w H co th et an R so co 3

3 Variation of HMW-GS Subunits at Glu-1

cy of oc

o (3 ef G 2 su h 7 2 cu re o sh th re 2 G (7 sh an d al g co D 2 Gl ibed above. Gels were run at 40mA/gels at room erature and stopped 30 min after the tracking dye had

ed the bottom of the gel. They were stained as ibed above.

uantification of Protein

rcentage of nitrogen was determined on 0.25 g of by the Dumas co

analyzer according to Approved

2000) and reported as protein by N*6.25. (American ciation of Cereal Chemists. 2000 approved method of

ACC, 9th ed. Method 38-12).

DS-Test

Five ml of distilled water put in the cylinder and of flour added to the cylinder.

closed and shaken 1

nd at the second minute; the cylinder was shaken 15 times (one per second). At the completion of 3 nd 45 second, the cylinder again was shaken 15 times per second) and 5 ml of SDS/ lactic acid [20 g of SDS of distilled water and 20 ml of mix (10 ml lactic acid and 80 ml of distilled water) was added to SDS on] added to cylinder and the cylinder was shaken 4 . At the completion 6, 8 and 10 minute, the cylinder haken again 4 times. The volume of sediment read at mpletion 25 min. The value obtained is multiplied by obtain a value of sedimentation volume compared 100ml of solution.

easurement of Quality Traits ousand Kernel Weight:

wheat grain by removing all dockage

broken seeds, and other foreign material. The samples (300 gram /variety) divided into five lots of 60 gram and the number of seeds was calculated for each lot.

• Length And Large Kernel Diameter: the diameter of kernel was calculated by taking several photos of five lots of each variety (10 gram/lot). The diameter was determinate by Image-J software.

• Flour Yield: five lots of 60 gram of each variety were e hted before and after grinding. Th

w ig e flour yield was

measured by the difference between the weight of kernel before grinding and the weight of flour yield.

2.6. Data Analysis Electrophoregrams fo

(1) or absence (0) of each band noted. Presence bsence of bands were entered in a binary data matrix.

(least significant difference) test was carried out a statistical package SPSS-PC, version 15. UPGM eighted Pair Group Method with Arithmetic) used for ruct the dendrogram.

cussion

3.1. Characterization of Wheat Varieties by Quality Evaluation

Thousand kernel weight, length diameter kernel, r diameter kernel, protein content, SDS-Test and SDS

ported between the kernel size and protein content 'Brien and Ronalds, 1984). The highest value for length ernel diameter was that of Tammuz, Aras and Rabia

hile Cham 4, Costantino and Cham 6 showed lower alue for length kernel diameter (Table 1). The varieties ham 4, Cham 6 and Tammuz had the highest values of rger kernel diameter and Aras, Costantino and Rabia had e lowest values. The flour protein content ranged from .14 to 9.24 and Costantino showed the lowest value.

ccording to the SDS-test, the varieties were divided into ve groups. The SDS-test of Tammuz was highly gnificant than the rest of varieties. The variety Cham 4 ad the lowest value of SDS-test suggesting poor insoluble rotein content. The SDS-sedimentation volume correlated

ith the amount of total HMWG subunits and individual MWG subunits. Some subunits were positively

rrelated, and the others were negatively correlated with e sedimentation volume (Seilmeier et al., 1991). Carrillo al. (1990) reported that HMWG subunits had additives

d epistatic effects on the SDS-sedimentation volume.

harrabti et al. (2003) studied the relationship between me quality traits and yield of durum under different nditions.

.2. Characterization of Wheat Varieties by SDS-PAGE .2.1. Allelic

The wheat varieties analyzed showed four different HMW glutenin banding patterns (Table 2). The frequen

currence of HMW glutenin subunits with composition f 1 (50%), 7+9 (16.67%), 7 (50%), 20 (33.33%), 5+10

3.33%) and 2+10 (66.67%). Subunit 20 had different fects on the functional properties in three subunits at the lu-D1 allele. Subunit 20 with a background of subunits +12 had a smaller negative effect than subunits 5+10 and

bunits 2.2+12 (Kanenori et al., 2003). Subunits 17+18 ad higher values of functional properties than subunits +9 with a background of subunits 2.2+12 (Kanenori et al., 003). On the other hand, based on the analysis of various

ltivars from several countries, Moonen et al., (1983) ported that the Glu-A1b alleles exerted stronger effects n the SDS sedimentation than Glu-A1a.

The varieties Tammuz, Aras and Costantino owed the pre ence of allele 1 at the locus Glu-A1. Ons e other hand, the varieties Rabia, Cham 4 and Cham 6 vealed the absence of allele at the locus Glu-A1 (Table ). All varieties showed the presence of alleles at locus lu-B1. At Glu-B1, there are three types of alleles a, c, e

, 7+9 and 20) (Tables 2 and 3). The SDS-PAGE analysis owed two types of alleles at locus Glu-D1: a, d (5+10 d 2+12) (Tables 2 and 3). Figure 2 showed the two imensional A-PAGE x SDS-PAGE maps of reduced and

kylated glutenin from wheat varieties. The HMW-GS of lutenin were identified easily (Figure 2). The results nfirmed the significantly beneficial effects of the Glu- 1d on the dough and gluten strength (Tadashi et al., 006). Kanenori et al. (2003) showed that the Glu-A1a and

u-A1b alleles exerted similar effects on the gluten score.

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© 2009 Jordan Journal of Biological Sciences. All rights reserved - Volume 2, Number 2 58

Table 1. Thousand kernel weight, length kernel diameter, large kernel diameter, flour yield, flour protein content, SDS- sedimentation volume and SDS index of wheat varieties.

Figure 1. SDS-PAGE gel showing the HMW and LMW-GS glutenins of bread wheat varieties.

1: Tammuz, 2: Aras, 3: Rabia, 4: Costantino, 5: Cham 4, 6: Cham

6. re:

al le: alleles encoded by Glu-

A

W glutenin

score sc h sc T v fo m P m v sp b fo ap cu (1 fr b A an el g K R L T n co fr co re es b et

Arrow head: alleles encoded by locus Glu-D3, arrow squa leles encoded by Gl-B3, arrow circ

TKW: Thousand kernel weight, LESD: Length kernel diameter, LASD: Large kernel diameter, FY: Flour yield, PC: Protein conten SDS-test: Sedimentation Test, SDSi: Sedimentation Index.

differe Table variet

3.

From the electrophoretic spectra the individual HM subunits were determined and so Glu-quality t,

Values shown by the same letter are not significantly nt (p=0.05) by LSD test.

2. HMW subunits composition and quality score of wheat ies.

was calculated (Table 2). The highest value of Glu- ore was achieved by the variety Tammuz. On the other and, Cham 4 and Cham 6 showed the lowest value of

ore quality (Table 2). These results show that the variety ammuz contains more of insoluble protein than others of arieties. The couple of subunits 5+10 is the good marker

r breading-making quality and the subunit 20 is the arker for weak wheat quality (Payne and Lawrence 1983, ayne et al., 1987a). HMW-GS may be used as a

olecular marker of bread-making quality of wheat. The erified correlations between bread-making quality and

ecific HMW subunit of glutenin can be utilized by wheat reeders using SDS-PAGE of proteins as a screening test

r the prediction of breading-making quality of wheat.

Several electrophoretic techniques have been pli

Table 3. Allele’s composition at the Glu-1 loci of bread wheat varieties.

ed to separate the subunits of glutenin in bread wheat ltivars. The method proposed by Gupta and Shepherd 990) consists of a two-step, one-dimensional actionation by SDS-PAGE in which glutenin is reduced efore being loaded onto a gradient gel for the second step.

great number of bread wheat cultivars have been alyzed by this method. Three different groups of ectrophoretic patterns were identified and attributed to enomes A, B and D. A similar approach was proposed by

helifi and Branlard (1992) and Redealli et al., (1995).

edealli (1995) showed the fraction of HMW-GS and MW-GS of glutenin by one and two dimensional gels.

he storage proteins of hexaploid wheat are important utritionally but, above all, because of the unique

hesive-elastic properties they bestow on dough made om wheat flours. The HMW subunits of glutenin are nsidered to be the most important components with spect to the baking quality. Correlations have been tablished between particular HMW glutenin subunits and read-making quality of wheat (Payne et al., 1987a; Kriac

al., 1997).

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Figure 2. Two-dimensional A-PAGE x SDS-PAGE fractionation of HMW and LMW-GS from bread wheat varieties. a: Costantino, b:

Tammuz, c: Rabia, d: Aras, e: Cham 6, f: Cham 4.

3.2.2. Allelic Variation of LMW-GS Subunits at Glu-3

Lo e

the varia qualit wheat profil gluten differ bands of the and C

SDS-PAGE gel also showed th with a

1). Ea subun The s the va the va Tamm and C

Table 4. Allele’s composition at the Gli-3 loci and LMW pattern of bread wheat varieties.

the other hand, the profile of LMW subunit C divides the v

A Ch PA lo en w molecular weight glutenins are important becaus

tion in LMW-B glutenin decides the end use y. We studied the LMW-GS composition of the six varieties by SDS-PAGE analysis. SDS-PAGE es of LMW glutenins showed five different LMW

in subunits at Glu-3 loci (Figure 1, Table 4). The ences between the patterns were for mobility of . The LMW-5 is the most frequent type and the rest m are present only in one variety. We detected the B subunit of LMW-GS in all varieties (Figure 1). The e C subunit of LMW-GS higher mobility than B subunit of LMW-GS (Figure ch variety had 5-6 bands of LMW glutenin B and C its coded Glu-A3, Glu-B3 and Glu-D3 loci (Table 4).

ame variations were detected in the LMW-Gs among rieties. According to the profile of LMW subunit B, rieties divided into four groups: group 1 contains uz, group 2 includes Rabia, group 3 contains Aras ostantino, group 4 includes Cham 4 and Cham 6. On

arieties into five groups: group 1= Tammuz, group 2=

ras, group 3= Rabia, group 4= Costantino, group 5=

am 4 and Cham 6. Several LMW subunits in the SDS- GE patterns are not attributed to any specific Glu-3 cus because of the overlapping between polypeptides coded by different alleles (Pogna et al., 1995). Low

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© 2009 Jordan Journal of Biological Sciences. All rights reserved - Volume 2, Number 2 60

molecular glutenin subunits are important in determining the dough viscoelastic properties of hexaploid and tetraploid wheat flours (Pogna et al., 1990). However, the basis of differences in effects of different low molecular weigh

unkno the G Glu-A differ both doug 3.3. G

In

HMW and LMW-GS was performed in order to analyze investigate genetic

electr differ 18 ba varia (Figu To bread

glute , phylogenetic trees were drawn from the alignmen

LMW storag PAGE amon that group Aras Cham the o other whea HMW 2005 estim becau qualit

contr the w the v high Ident

in Iraq may be useful for selection aims in breeding programs to determine the relationship between gluten visco-elastic properties.

A

The author thanks the agriculture department of

S ing the seeds and LNCV and Unità

di ricerca per la maiscoltura,Via Stezzano, 24, Bergamo, Italy

su

R

A dean ML.1994. Characterization of the 1B/1R

tr extractable protein concentrate.

E A

m th ed. Method 38–12A Wet gluten, dry

gl ican

A

C e

of ons of

hi tr G

C .

Th glutenin sub

po cu t subunits alleles on dough properties is still largely wn. Gupta and MacRitchie (1991) have shown that lu-A3m produces no major B subunit, whereas allele 3a codes for the major B subunit. This allelic ence was found to be responsible for variation in the size distribution of the glutenin polymers and h strength.

enetic Diversity among The Varieties

this study SDS-PAGE of grain storage proteins molecular weight of gluten subunits and

diversity among different wheat varieties. The ophorogram showing proteins banding pattern of ent wheat varieties are given in Figures 1. A total of nds were obtained among which 12 bands were show tion but the other bands common in all varieties

re 1).

investigate evolutionary relationships among the wheat varieties according to HMW and LMW nin subunits

t of these varieties based on both HMW and -GS (Figure 3). Cluster analysis of wheat grain e proteins was performed on the results of SDS-

using the software UPGMA to find out the diversity g the given wheat varieties. The Alignment indicated the phylogenetic tree was divided into three parts:

1= Cham 4, Cham 6, Rabia; group 2= Tammuz, and group 3= Costantino. The varieties Cham 4 and 6 showed more similarity than others varieties. On ther hand, Costantino showed more distance with the s varieties. Genetic diversity of European spelts t was evaluated by constructing the dendrogram for

and LMW glutenin subunit bands (Xueli et al., ). Fufa et al. (2005) reported that the genetic diversity

ates based on seed storage protein were lowest se they were the major determinants of end-use y, which is a highly selected trait.

Figure 3. Dendrogram of bread wheat varieties showing the

relatio d

LMW-

In conclusion, the results showed that there was the significa

quali HMW whea

nship among the varieties based on SDS-PAGE-HMW an GS.

nt difference among the varieties for some of ty traits tested. The electrophorogram revealed that subunit 7+9 which related with the strong quality of t for baking is less frequent in the varieties tested. In ast, HMW subunits 2+12 and 20 which related with eak quality for making of bread is more frequent in arieties tested. The LMW-B subunits revealed more level of polymorphisms than LMW-C subunits.

ification of glutenin subunits in bread wheat varieties

cknowledgements

ulaimanyh for provid

especially Dr. Rita REDAELLI for help and pporting the work

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References

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